Microorganisms capable of metabolizing cholesterol are potential sources of enzymes useful in an enzymatic assay of cholesterol in complex mixtures such as blood serum, etc. This is particularly so if the microorganisms can use cholesterol as a sole carbon source, for in this assay process cholesterol must be degraded by oxidative enzymes.
Stadtman, T. C., Methods in Enzymology, Vol. 1, Colowick, S. P. and Kaplan, N. O., Eds. Academic Press, N.Y. 1955, P. 678 and Stadtman, T. C., Cherkes, A. and Anfinsen, J., Biol. Chem., 206, 510 (1954) reported the preliminary purification of an enzyme from Nocardia cholesterolicum, an organism originally isolated by Schatz et al (Schatz, A., Savard, K., and Pintner, I. J., J. Bacteriol., 58, 117-125 (1949). Stadtman's enzyme, "cholesterol dehydrogenase," was purified sufficiently for use in a cholesterol assay based on the measurement of the increase in absorbance at 250 nm owing to the formation of cholest-4-en-3-one. Since as we have now determined, the direct acceptor of cholesterol electrons in this oxidation is oxygen, the enzyme should properly be called cholesterol oxidase according to current convention.
The bacterial strains described by Stadtman when cultured as described in the aforementioned references produce very low enzyme levels which are not practical for commercial operations. These levels are sufficiently low that purification of the enzyme is a very remote possibility for achieving a commercial operation.
Goodhue et al in U.S. Pat. No. 3,909,359 issued Sept. 30, 1975, describe an improved method for the production of the Stadtman cholesterol oxidase which comprises the steps of:
A. growing the bacterium Nocardia cholesterolicum species NRRL 5767 or NRRL 5768 in a medium in which cholesterol or a suitable derivative thereof serves as an auxiliary source of carbon; and PA1 B. isolating from said medium a cell-free extract containing the active enzyme.
Although the method described in this application is greatly improved over the original synthesis described by Stadtman and can be said to render the process commercially practical, it still poses a shortcoming in that the enzyme is produced predominantly intracellularly. For this reason actual extraction of the enzyme from the growth medium requires the use of timeconsuming, expensive cell disruption techniques such as homogenization, etc.
German OLS 2,246,695 published Mar. 29, 1973, describes a method for isolating a cholesterol oxidase enzyme produced by a culture of Nocardia microorganism identified as NRRL 5635 and NRRL 5636. According to the method described therein, the harvested cells are treated with a nonionic surfactant and stirring at room temperature to release a large proportion of the enzyme from the cells into the supernatant thereby eliminating the need for involved cell extraction and isolation techniques. Using this technique for enzyme extraction we have calculated that yields on the order of about 40 to 160 U per liter are obtained.
Reese, E. T. and Maquire, A., in Surfactants as Stimulants of Enzyme Production by Microorganisms, Applied Microbiology, February, 1969, P. 242-245 describe the observation that the addition of sorbitan polyoxyethylene monooleate (Tween 80 from Atlas Chemical Co., Wilmington, Delaware) and other nonionic surfactants to fungal cultures which normally produce extracellular enzymes, results in a marked increase in enzyme yield.
British Pat. No. 1,385,319 describes a process for producing cholesterol oxidase from Nocardia species NRRL 5635 and NRRL 5636. During the fermentation, a suspension of cholesterol is slowly added as an inducer for cholesterol oxidase. A non-ionic surfactant, Tween 80, at a level of 3% by volume is used to disperse the cholesterol in the suspension. This results in only a minute amount of non-ionic surfactant in the fermentation medium, which amount is apparently insufficient to produce extracellular enzyme.
The bacterial cultures known to produce cholesterol oxidase, normally produce the enzyme intracellularly. The present invention provides an improved method for the production of cholesterol oxidase, wherein the enzyme is produced extracellularly thereby eliminating the need for cell disruption.